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Upregulation of the zebrafish Nogo-A homologue, Rtn4b, in retinal ganglion cells is functionally involved in axon regeneration.

Welte C, Engel S, Stuermer CA - Neural Dev (2015)

Bottom Line: MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner.With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side).The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. Cornelia.welte@uni-konstanz.de.

ABSTRACT

Background: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals.

Results: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO.

Conclusions: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

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Upregulation of Rtn4b in zebrafish RGCs after ONS. RGCs in retina whole mounts showed weak immunostainings in the cytoplasm after exposure to Rtn4b AB (A). (B,E,H) Labeling with Phalloidin against F-actin shows all cells and their cytoplasm. (C,F,I) Merge of (A,B), (C,D), and (G,H) with DAPI stainings to visualize nuclei. (D,E,F) 5 days after ONS, the RGCs exhibit a significant increase in size and increase in Rtn4b labeling intensity (48% in comparison to control, P < 0.01) in the cytoplasm. (G,H,I) 10 days after optic nerve sections, the size on the RGCs and the intensity of Rtn4b staining is still highly increased (53% over controls, P < 0.01). Scale bar, 10 μm. (J) This apparent increase in the 100 kd Rtn4b protein is also seen in Western blots with retinae at 5 (P < 0.05) and 10 days (P < 0.0001, Student’s T-test) after ONS. Anti-alpha tubulin served as loading control.
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Fig3: Upregulation of Rtn4b in zebrafish RGCs after ONS. RGCs in retina whole mounts showed weak immunostainings in the cytoplasm after exposure to Rtn4b AB (A). (B,E,H) Labeling with Phalloidin against F-actin shows all cells and their cytoplasm. (C,F,I) Merge of (A,B), (C,D), and (G,H) with DAPI stainings to visualize nuclei. (D,E,F) 5 days after ONS, the RGCs exhibit a significant increase in size and increase in Rtn4b labeling intensity (48% in comparison to control, P < 0.01) in the cytoplasm. (G,H,I) 10 days after optic nerve sections, the size on the RGCs and the intensity of Rtn4b staining is still highly increased (53% over controls, P < 0.01). Scale bar, 10 μm. (J) This apparent increase in the 100 kd Rtn4b protein is also seen in Western blots with retinae at 5 (P < 0.05) and 10 days (P < 0.0001, Student’s T-test) after ONS. Anti-alpha tubulin served as loading control.

Mentions: Next, we analyzed changes in axotomized RGCs in retina whole mounts (Figure 3A, B, C, D, E, F, G, H, I). RGC somata at 5 days after ONS increased in area by 87% (the cell body reaction), and the cytoplasm was filled entirely by Rtn4b AB labeling (Figure 3D) associated with cloudy structures, typical for ER staining with anti-protein disulfide isomerase (PDI) in mammalian cells (ABCAM home page). Rtn4b staining intensity was upregulated in intensity by 48% when compared to controls (P < 0.01). Ten days after ONS, Rtn4b protein levels were even more elevated with an increase of 54% in comparison to control (P < 0.01).Figure 3


Upregulation of the zebrafish Nogo-A homologue, Rtn4b, in retinal ganglion cells is functionally involved in axon regeneration.

Welte C, Engel S, Stuermer CA - Neural Dev (2015)

Upregulation of Rtn4b in zebrafish RGCs after ONS. RGCs in retina whole mounts showed weak immunostainings in the cytoplasm after exposure to Rtn4b AB (A). (B,E,H) Labeling with Phalloidin against F-actin shows all cells and their cytoplasm. (C,F,I) Merge of (A,B), (C,D), and (G,H) with DAPI stainings to visualize nuclei. (D,E,F) 5 days after ONS, the RGCs exhibit a significant increase in size and increase in Rtn4b labeling intensity (48% in comparison to control, P < 0.01) in the cytoplasm. (G,H,I) 10 days after optic nerve sections, the size on the RGCs and the intensity of Rtn4b staining is still highly increased (53% over controls, P < 0.01). Scale bar, 10 μm. (J) This apparent increase in the 100 kd Rtn4b protein is also seen in Western blots with retinae at 5 (P < 0.05) and 10 days (P < 0.0001, Student’s T-test) after ONS. Anti-alpha tubulin served as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374419&req=5

Fig3: Upregulation of Rtn4b in zebrafish RGCs after ONS. RGCs in retina whole mounts showed weak immunostainings in the cytoplasm after exposure to Rtn4b AB (A). (B,E,H) Labeling with Phalloidin against F-actin shows all cells and their cytoplasm. (C,F,I) Merge of (A,B), (C,D), and (G,H) with DAPI stainings to visualize nuclei. (D,E,F) 5 days after ONS, the RGCs exhibit a significant increase in size and increase in Rtn4b labeling intensity (48% in comparison to control, P < 0.01) in the cytoplasm. (G,H,I) 10 days after optic nerve sections, the size on the RGCs and the intensity of Rtn4b staining is still highly increased (53% over controls, P < 0.01). Scale bar, 10 μm. (J) This apparent increase in the 100 kd Rtn4b protein is also seen in Western blots with retinae at 5 (P < 0.05) and 10 days (P < 0.0001, Student’s T-test) after ONS. Anti-alpha tubulin served as loading control.
Mentions: Next, we analyzed changes in axotomized RGCs in retina whole mounts (Figure 3A, B, C, D, E, F, G, H, I). RGC somata at 5 days after ONS increased in area by 87% (the cell body reaction), and the cytoplasm was filled entirely by Rtn4b AB labeling (Figure 3D) associated with cloudy structures, typical for ER staining with anti-protein disulfide isomerase (PDI) in mammalian cells (ABCAM home page). Rtn4b staining intensity was upregulated in intensity by 48% when compared to controls (P < 0.01). Ten days after ONS, Rtn4b protein levels were even more elevated with an increase of 54% in comparison to control (P < 0.01).Figure 3

Bottom Line: MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner.With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side).The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. Cornelia.welte@uni-konstanz.de.

ABSTRACT

Background: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals.

Results: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO.

Conclusions: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

Show MeSH
Related in: MedlinePlus